I have spent my four most
productive years in Jeff Corden’s lab and developed most of my scientific
views and approaches by working hard on two projects aimed to understand
the function of the C-terminal
domain of eukaryotic RNA polymerase II (CTD).
My
first project was to address CTD function using methods of yeast genetics.
Corden’s lab was competing with Rick Young’s lab at MIT. R. Young's
group has shown that the deletion of 18 out of 25 CTD repeats in yeast
polymerase II cause the cold sensitive phenotype and that the deletion
of 19 CTD repeats is lethal. Marilyn
West in Corden’s lab has constructed CTD substitution mutants in which
putative CTD phosphorylation sites were substituted for alanines to mimic
dephosphorylated state or glutamic acids to mimic phosphorylated state
(YSPTSPS to YA/EPTSPS
and YSPTSPS to YSPTA/EPS mutants).
She also found that such substitutions are lethal and yeast can not live
with such mutated CTDs.
My goal was to find the
mutant yeast cells that actually can grow with such substituted CTDs by
acquiring the compensatory (suppressing) mutations. I have
isolated such suppressors during my rotation in Jeff Corden's lab and came
back to carry out my Ph.D. thesis in his lab. The suppressor mutants
were extremely slow growing (colony were visible only after 5-7 days of
growth as opposed to 2 days for wild type S. cerevisiae strain).
The technique that I used for cloning the suppressor by genetic complementation
involved the plasmid
shuffling method developed in Jeff
Boeke’s lab. That is why, Jeff Boeke has contributed even more
to this project than Jeff Corden himself.
In spite of Jeff Boeke’s
warning to not work with slow growing yeast mutants, I went ahead to clone
a suppressor gene from one of the mutants. As a result of my effort
I isolated a novel yeast gene with unknown function. In parallel
with my work R. Young’s graduate student at MIT cloned the suppressor of
cold sensitive CTD deletion. As it turned out he cloned the same
gene. R. Young named this new gene SRB9 and I called
it SCA1 (GB#
P38931). I have also found that SCA1 gene was non-essential
for yeast viability and that
SCA1 deletion is enough to suppress
lethal CTD substitutions. This fact suggests that SCA1
suppression is a bypass effect. SCA1 deletion relives or compensates
for the “weak” performance of the CTD mutants. I, personally, believe
that most of R. Young’s SRBs, that he found as suppressors of cold sensitive
CTD mutation, are also bypass mutants. In fact it is much easier
for yeast to bypass cold sensitivity than lethality and that is why he
found so many SRBs. I also think that his “holoenzyme
hypothesis” is complete nonsense and total waste of tax-payers money.
(I am trying to be polite here).
Please, read references
on myself written by Jeff Boeke and Jeff
Corden and P. Hieter, who was a member of my
thesis committee, upon my graduation.